CN113558012A

CN113558012A - Method for breeding female multiple births by using mammals

Info

Publication number: CN113558012A
Application number: CN202110898713.4A
Authority: CN
Inventors: 陈米米
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-10-29

Abstract

The invention provides a method for breeding female multiple fetuses by mammals, and relates to the technical field of mammal breeding. The method comprises the following steps of selecting female ovum female parent of mammal with chromosome XY of deletion SRY gene and male sperm of mammal with polyembryony gene as male parent, inseminating the male parent sperm and the female parent ovum in vitro to obtain offspring containing polyembryony gene and deletion SRY gene, crossing the offspring to obtain female individual with chromosome XY of expression polyembryony character and SRY gene deletion character.

Description

Method for breeding female multiple births by using mammals

Technical Field

The invention relates to the technical field of mammal breeding, in particular to a method for breeding female multiple fetuses by mammals.

Background

The multiparous breeding of mammals is an industry problem which needs to be solved urgently at present, only the multiparous breeding of sheep is stable at present, and the multiparous breeding of other mammals such as cattle and the like still needs to be solved at present. According to various research results related at present, the sheep can be bred in multiple fetuses, mainly because of the expression of the multiple fetuses genes in the sheep, and other mammals such as cattle and the like have relatively rare individuals with the multiple fetuses genes expression and are not easy to inherit.

In addition, in mammals such as cattle, sheep and the like, the milk yield of mainly ewes and cows is higher, the skin and hair of the cows and ewes are smoother, and the utilization value is higher, so that the direct utilization value of females in the mammals is obviously higher than that of males, and how to stably breed more females in the mammals is a technical problem which needs to be solved at present.

Disclosure of Invention

The invention aims to provide a method for breeding female multiple fetuses by mammals, which not only can enable the mammals to breed multiple fetuses, but also can enable the bred offspring to be almost female, thereby improving the utilization value of the mammals, enabling the breeding of the mammals to be easier, and using an artificial insemination mode for the mammals, not only leading the operation to be simpler and more convenient, but also greatly shortening the breeding time.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

A method for breeding female multiple fetus by mammal includes such steps as choosing female ovum of female mammal whose chromosome is XY and male sperm containing multiple fetus gene, inseminating the male sperm with female ovum to obtain the filial generation containing multiple fetus gene and missing SRY gene, and crossing the filial generations to obtain the female individual whose chromosome is XY and able to express multiple fetus character and SRY gene missing character. The SRY gene, also known as a testis determinant, is a sex-determining gene of a male, and refers to a gene fragment on the Y chromosome that specifically determines the sex of the male of an organism. The human SRY gene is located in Yp11.3, contains only one exon, has no intron, has a transcription unit length of about 1.1kb, and encodes a protein of 204 amino acids. Since the SRY protein contains a typical DNA binding domain: the high mobility class non-histone box motifs resemble known transcription factors, so it is assumed that SRY encodes a transcription factor. The HMG domain of SRY binds to DNA in a sequence-specific manner, introducing a sharp turn in the double helix structure. There is evidence that mutations in the HMG domain of patients with sexual inversion can be divided into two classes: affecting DNA binding and affecting DNA bending, suggesting that both properties are important for the SRY protein to perform transcriptional regulatory functions. HMG domains have been found to bind calmodulin with high affinity in vitro. The functional significance of this phenomenon is unclear, but suggests that the activity of SRY may be regulated by another level. The human SRY gene is positioned on the short arm of a male Y chromosome, is about 850bp long and only contains a single exon, and contains a poly A tail, two transcription starting sites and an open reading frame in the middle; the SRY gene encodes a protein of 204 amino acids, called SRY protein, which contains a highly conserved region of 79 amino acids at one end, the hyper-phoretic box motif, which has a high degree of homology in different mammals. For example, human and bovine SRY genes have HMG box homologies as high as 84%, and numerous studies have demonstrated that the normal development of the male reproductive organs in mammals is premised on the normal functioning of the SRY genes. And the function of the SRY gene has time sequence specificity, and the methylation degree of the SRY gene is obviously different in the development process of male embryo and fetus, namely different stages of the generation of male reproductive organs.

Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:

the embodiment of the invention provides a method for breeding female multiple fetuses by mammals, which can not only enable the mammals to breed multiple fetuses, but also enable the bred offspring to be almost female by hybridizing multiple fetuses and a mammal group lacking SRY genes, thereby improving the utilization value of the mammals and enabling the breeding of the mammals to be easier.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

The embodiment of the invention provides a method for breeding female multiple-fetus by mammals, which comprises the following steps of selecting a mammalian female without multiple-fetus genes as a first female parent and a mammalian male with multiple-fetus genes as a first male parent, hybridizing the first male parent and the first female parent to obtain an F1 filial generation containing multiple-fetus genes, crossing the F1 filial generation to obtain an F2 filial generation containing multiple-fetus genes, using the F2 filial generation as a second male parent, selecting the mammalian female with XY chromosome and missing SRY genes as a female parent, hybridizing the female parent and the second male parent to obtain an F3 filial generation containing multiple-fetus genes and missing SRY genes, crossing the F3 filial generation for multiple generations, and screening out individuals with multiple-fetus genes and missing SRY genes in the filial generation to obtain the mammalian for breeding multiple-fetus by females.

In some embodiments of the invention, the female parent further comprises an activated SOX9 gene. The SOX9 gene is normally only active in men, and when the SOX9 of men is turned on, the activity of FOXL2 is inhibited and thus stops for life. This is the opposite in a female, and FOXL2 will be activated first. Although FOXL2 is widely known in the academia to be important for women to maintain female characteristics and the growth of ovaries, scientists do not expect ovulatory cells in the ovaries to be absorbed by the SOX9 gene and thereby perform male fertility. The Sox9 gene is another sex-determining Sox family member found after the Sry gene and is a transcription activator, and is located on an autosome. Human Sox9 contains two introns and is highly conserved at Sox9 in mammals, birds and reptiles 8. Human Sox9 gene mutation can cause bone malformation syndrome; XX individuals with additional Sox9 developed males, while most XY individuals with only one functional Sox9 developed females or sexes, suggesting that Sox9 plays an important role in either skeletal development or in sex determination. The mouse Sox9 gene is only expressed in male genital gnawing, and the expression time is later than that of Sry. The anti-mullerian hormone gene is an important gene at the downstream of Sox9 and codes the anti-mullerian hormone in early male development of vertebrates, and the Sox9 gene plays a key role in activating Amh: soX9 protein is combined with Amh promoter to promote the expression of Amh gene. The SOX9 protein and the SRY protein have similar functions, and under the premise of Sry gene deletion, Sox9 can also induce male sex differentiation so as to ensure the normal development of male gonads.

In some embodiments of the invention, the female parent further comprises an activated SOX3 gene. Sox3 located on the X chromosome the mouse segment most conserved in the X chromosome is a gene related to Sry. Sox3 is the gene with the highest similarity with Sry in the Sox gene family, Sry is the inhibitor of Sox3, and 8 is formed by truncation of Sox3 flanking sequences. Sox3 was therefore considered to be a nasal progenitor to the evolution of Sry, and most scholars considered that both could also be a pair of alleles.

In some embodiments of the invention, the female parent further comprises an activated DAX1 gene. The Daxc1 protein is 1 protein of the nuclear hormone receptor superfamily encoded by the Dax1 gene (DSS-AHC criticalsegion on the X chromoe gene 1), which was discovered in XY female patients in 1980 and the gene was cloned in 1994 [6 ]. Daxl protein is a negative regulator of transcription regulated by retinoic acid receptors. It is shown that when a mutation in a certain position of human X chromosome is doubled, the segment of the mutation site which can cause XY individuals to develop towards female direction is called sex reversal gene, and said gene and congenital hypogonadism gonadotropin hyposecretion gonadal dysfunction are linked together on X chromosome. The human Dax1 gene encodes a nuclear protein that binds to DNA and regulates its transcription, and in general, SRY inhibits the Daxl gene but causes the normal XY sexual reversal of SRY into females when extra active copies of Daxl are present. The appearance of an extra Daxl copy in male mice will result in sexual inversion: when a DNA fragment carrying the Dax-1 gene is introduced into XY mouse embryos, testicular development retardation and even sexual inversion occur, which shows that the Dax1 gene has an inhibiting effect on the Sry gene. DAxl is an anti-testosterone that prevents the reproductive kurtosis from differentiating into males by inhibiting the synergistic effect of steroidogenic factor 1 and wilm's tumor suppressor 1 in sexual differentiation. Studies have shown that Dax1 can disrupt Wtl function to inhibit Sry expression while inhibiting transcriptional activation via nuclear receptor co-inhibitors. Both Daxl and Sry products are antagonistic to each other, and an increase in Dax1 expression results in the development of the individual towards females whereas an increase in Sry expression induces the development of the individual into males.

In some embodiments of the invention, the female parent further comprises an activated SF1 gene. sf1, also known as Ad4BP or NRSA1, is a family member of the orphan hormone receptor, whose cDNA closely resembles the cDNA derived from a library of cancer cell eDNA in mice. Sf1 has obvious DNA binding region and ligand binding region, and its action is mainly expressed in the following two aspects: (1) one of the determinants of the adrenocortical hormone expression regulation was found by examining tissues of adult mice, and as long as the expression activity of Sf1 was also expressed in podocytes, it was found that Sf1 plays an important role in adrenal development, steroid production and early embryonic development. (2) sf1 directly regulates the expression of the mullerian inhibitor gene. Early embryo podocyte transfection shows that Sf1 and a regulatory region Mis-RE-1 of Mis can interact with each other to activate, and also shows that Sf1 and Mis of other cell lines do not express early activity, Sf1 ligand which is dependent on Mis transcription is probably contained in podocytes, the ligand is combined with the ligand binding region to cause the ligand binding region to be separated from Sf1, and the Sf1 activates the expression to promote the transcription of Mis. Sf1 also binds to the promoter of Daxl gene to induce its transcription.

In some embodiments of the invention, the parent further comprises a deletion in the AMH gene. Amh, also known as Mis, is an important factor in male sexual differentiation, and mutations in this gene lead to permanent Muller tube syndrome. The expression of the Amh gene begins rapidly after Sry expression as the first identifiable product in the supporting cell. Studies have shown that the Sry protein does not bind directly to the regulatory elements of Amh, but rather induces Ahm expression in an indirect manner, suggesting that other intermediate factors should be involved between Sry and Amh. Then, it is found that the mutation of the binding site of the Amh gene on the binding sequence 1 with Sox9 protein and S1 at the upstream of the Amh promoter leads to the reduction of the expression quantity of the Amh gene, and the mutation of the binding site on Sox9 leads to the complete disappearance of the expression quantity of the Amh gene, which indicates that Sox9 is probably an activation switch of the Amh gene, and once the Amh gene is activated, the Sf1 protein and the 1 protein jointly act to promote the expression of the Amh gene to improve the transcription level.

In some embodiments of the invention, the female parent further comprises a deletion of the WT1 gene. The wt1 gene inhibits cell division and differentiation and is related to the formation of spermary. The W1 gene plays an important role mainly in the development of the urogenital tract. Mice that knock out Wt1 gene lack male gonads and develop abnormal development, indicating that Wtl gene has a certain regulatory role in the development of male gonads. The human Wt1 gene is located on chromosome 17, and the DNA binding domain of Wt1 protein is 4 Cys-Cys zinc fingers. This gene mutation will lead to the Denys-Drash syndrome characterized by Wilms' tumor, renal and gonadal dysplasia.

In some embodiments of the invention, the parent further comprises a deletion of DMRT1, DMRT2 and DMRT3 genes. The protein encoded by the dmrt gene contains a conserved motif, the DM domain, which has DNA binding capacity. The number of 8 Dmrt family members reported so far, namely Dmrt, 2, 3, 4, 56, 7, 8, among them, Dmr2 has 2 subtypes, Dmrt2a and Dmrt2b, and other members except Dmrt8 only encode DM domains with high conservation. The existence of a homologue of Dmrt in various species is the only one of the sex-determining genes discovered so far that is conserved among species, and Dmrt1, 2 and 3 in human embryos have an important role in normal development of the testis, wherein Dmrtl is similar to Sry, and the deletion of this gene leads to the occurrence of sexual inversion. Current studies have shown that Dmrt1 is not a major gene for male sex determination, although it is involved in the differentiation of mammalian male gonads.

In some embodiments of the invention, the maternal host further comprises an activated WNT4 gene. The Wnt4 gene inhibits the differentiation of testicular interstitial cells, induces the formation of female ovaries, and over-expresses the Wnt4 gene to further up-regulate the expression level of DAX1, so that female phenotype is formed. When the Wnt4 gene of female rat is mutated, its ovary becomes masculinized, and interstitial cells secrete androgen, and further the middle renal duct is differentiated and the mullerian duct is deleted. The WNT/RSPO signal in conjunction with FOXL2 determines the development of the male gonads of mammals into female sex organs. The inhibition of the beta-catenin signal plays an important role in the development of male Scrtioli cells. Over-expressing CTNNB1 in Scrto-li cells, Sertoli cells differentiated into granule-like cells, and CTNNBI caused Fox12 expression by binding to Tcl/Lef specific sites in the promoter region of FOXL 2; over-expression of Foxl2 in Sertoli cells, however, resulted in the specific down-regulation of Sertoli cell-associated genes.

In some embodiments of the invention, the XY chromosome female polyembryony trait and the SRY gene deletion trait described above, which express both the polyembryony trait and the SRY gene deletion trait, are capable of stable inheritance. The stable inheritance of the polyembryony character and the female character can be ensured through stably inherited individuals.

The features and properties of the present invention are described in further detail below with reference to examples.

Examples

Cattle are taken as an example. 142 adult Guizhou Holstein cattle which are free of diseases, have the weight of not less than 60 kilograms, the body length of not less than 60cm and the body height of not less than 60cm and 58 adult Guizhou Holstein cattle which are free of diseases, have the weight of not less than 40 kilograms, the body length of not less than 40cm and the body height of not less than 40cm are taken as the first female parent. Taking 127 adult Shandong European wild cattle which are not diseased, contain FecB and SRY gene deletions, SOX9, SOX3, DAX1, SF1, AMH gene deletions, WIT1 gene deletions, DMRT1, DMRT2 and DMRT3 gene deletions and WNT4 gene, wherein the adult Shandong European wild cattle which are not less than 70 kg in body weight, not less than 80cm in body length and not less than 80cm in body height, 73 adult European wild cattle which are not diseased, contain FecB and HGTP genes, not less than 50 kg in body weight, not less than 70cm in body length and not less than 70cm in body height as a first male parent; artificial insemination is carried out to give sperms of wild cattle in Shandong Europe to ova of Guizhou Holstein cattle, fertilized ova after the embryos are developed are sent to the uterus of the Guizhou Holstein cattle for culture, after three years, offspring are screened out to be adult bulls which are free of diseases, contain FecB and HGTP genes, have the weight of not less than 90 kilograms, have the body length of not less than 80cm and have the body height of not less than 80cm, adult cows which are free of diseases, contain FecB and HGTP genes, have the body weight of not less than 70 kilograms, have the body length of not less than 70cm and have the body height of not less than 60cm, adult cows which are free of diseases, contain FecB and HGTP genes, have the body length of not less than 50cm and have the body height of not less than 40cm, F1 offspring is obtained, and then the offspring of the whole year old cattle are naturally mated with the offspring F1, obtaining and screening offspring after three years, and obtaining F2 offspring 815 of the whole year old cows which are not sick, contain FecB and HGTP genes, have the weight of not less than 90 kilograms, the body length of not less than 80cm and the body height of not less than 80cm, have no sick, contain FecB and HGTP genes, have the weight of not less than 70 kilograms, the body length of not less than 70cm and the body height of not less than 60cm, have no sick, contain FecB and HGTP genes, have the weight of not less than 50 kilograms, the body length of not less than 50cm and the body height of not less than 40 cm.

Test example 1

Taking 50 of the F2 offspring adult cows and bull cows obtained in the example, performing natural mating, counting the number of nests of born cows after two years, counting the number of nests of the born cows, wherein the nests are multiparous (i.e., one nest of cows is 3 or more and has no dead fetus), and calculating the multiparous rate (the number of nests of multiparous cows/the total number of nests of born cows × 100%), obtaining the results shown in Table 1, taking 50 of the adult cows and bull cows of Hostan cows of Guizhou, performing natural mating, wherein the nests of multiparous cows (i.e., one nest of cows is 3 or more and has no dead fetus), and calculating the multiparous rate (the number of nests of multiparous cows/the total number of nests × 100%) after two years, obtaining the results shown in Table 2.

TABLE 1 Multi-fetus statistics for the F2 progeny

Total litter size of newborn cattle	Nest number of multi-fetal calf	Multiple rate of births
			133 nest	97 nest	72.93％

TABLE 2 multiparous statistical table of Hostan cattle offspring

Total litter size of newborn cattle	Nest number of multi-fetal calf	Multiple rate of births
			89 nest	26 nest	29.21

According to the results in tables 1 and 2, the nests of F2 offspring obtained in the example are far higher than those of Holstein cattle in Guizhou, and the multi-birth rate of the F2 offspring is also far higher than that of Holstein cattle, so that the multi-birth breeding rate of the new variety of yellow cattle cultured by the invention is obviously improved.

Test example 2

The 5 generations of Guizhou Holstein cattle and F2 which were born at the same time on the same day, were each placed under the same environment and were bred, wherein the Guizhou Holstein cattle, numbered a-E, and the F2 progenies, numbered A-E, were fed daily with the same and equal amount of feed, were fed daily with the same amount of clear water, were weighed and recorded daily, and the weights were compared after 30 days of culture, with the results shown in Table 5.

TABLE 5 weight control Table

As shown in the results in table 5, the development speed of the F2 progeny provided by this example is significantly higher than that of the common bostanus guizhou, so it can be concluded that the growth speed of the new variety of cattle provided by the present invention is almost the same as that of the common cattle.

Test example 3

Taking 50 of the F2 offspring adult cows and male bulls obtained in the example, naturally mating each other for two years, counting the number of nests of born cows, the number of nests of female cows therein, and counting the sex, the results are shown in Table 1, taking 50 of the adult cows and male bulls of Hostan cows, Guizhou, naturally mating each other for two years, counting the number of nests of born cows, the number of nests of female cows therein, and calculating the female rate, the results are shown in Table 2.

TABLE 3 Multi-fetus statistics for the F2 progeny

Total litter size of newborn cattle	Female cattle nest number	Multiple rate of births
			133 nest	119 pit	89.47％

TABLE 4 multiparous statistical table of the offspring of Holstein cattle

Total litter size of newborn cattle	Female cattle nest number	Multiple rate of births
			89 nest	42 nest	47.19％

According to the results in tables 3 and 4, the female litter size of the young cattle obtained by the F2 offspring obtained in the example is much higher than that of the young cattle of the Holstein cattle in Guizhou, so that the female fertility rate of the new variety of the cattle cultured by the invention is obviously improved.

Test example 4

Taking 50 adult and adult male horses of F2 offspring for natural mating, counting the number of nests of born horses after two years, counting the number of nests of multi-births (multi-births, namely one nest of horses is more than or equal to 3 and has no dead births) in the nests of the horses, calculating the multi-births rate (the number of nests of multi-births/the total number of nests of born horses multiplied by 100%), obtaining the result shown in Table 5, taking 50 adult female horses and 50 adult male horses of Holstein horses of Guizhou for natural mating, counting the number of nests of born horses after two years, obtaining the number of nests of multi-births (multi-births, namely one nest of horses is more than or equal to 3 and has no dead births) in the horses, calculating the multi-births rate (the number of nests of multi-births/the total number of nests multiplied by 100%), and obtaining the result shown in Table 6.

TABLE 5F 2 Multi-fetus statistical Table of descendants

Total litter size	Nest number of multi-baby horse son	Multiple rate of births
			96 nest	71 nest	73.96％

TABLE 6 multiple-birth statistical table for the offspring of common horses

According to the results in tables 5 and 6, the F2 offspring has litter size far higher than that of the common horses, and the miscarriage rate is also far higher than that of the common horses, so that the miscarriage rate of the new horse variety bred by the method is obviously improved.

Test example 5

Taking 50 adult elephants and adult elephants of F2 offspring for natural mating, counting the number of nests of the born elephants after two years, and counting the multi-births (the multi-births, namely one nest of elephants is more than or equal to 3 and has no dead births) of the elephants, and calculating the multi-births rate (the number of nests of the multi-births of the elephants/the number of nests of the total born elephants multiplied by 100%), obtaining the results shown in table 7, taking 50 adult female elephants and 50 adult elephants of the Hostan elephants for natural mating, and counting the number of nests of the born elephants after two years, and obtaining the multi-births (the number of nests of the multi-births, namely one nest of the elephants is more than or equal to 3 and has no dead births) of the elephants, and calculating the multi-births rate (the number of nests of the multi-births of the elephants/the number of total born elephants multiplied by 100%), wherein the results are shown in table 8.

TABLE 7 MULTI-TYRE STATISTS OF F2 SUSPENSION

Total elephant cub nest number	Elephant with multiple birthsNumber of cub nest	Multiple rate of births
			55 nest	33 nest	60.00％

TABLE 8 multiple-birth statistical table for descendants of common elephant

Total elephant cub nest number	Nest number of large elephant cubs with multiple fetuses	Multiple rate of births
			38 nest	8 nest	21.05％

According to the results in tables 7 and 8, the nest number of the elephant born by the F2 offspring is not only much higher than that of the common elephant, but also the multi-birth rate is much higher than that of the common elephant, so that the multi-birth rate of the new variety of the elephant cultivated by the invention is obviously improved.

In summary, embodiments of the present invention provide a method for breeding female multiple births by a mammal, which can cross a group of mammals with multiple births and missing SRY genes, so that the mammals can breed multiple births, and the bred offspring are almost female, thereby improving the utilization value of the mammals and facilitating the breeding of the mammals.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A method for breeding female polyembryony of mammal includes such steps as choosing female ovum of mammal with XY chromosome and male sperm containing polyembryony gene, inseminating the male sperm with female ovum to obtain the filial generation containing polyembryony gene and SRY gene, and crossing the filial generation to obtain the female individual with XY chromosome, which can express the polyembryony character and SRY gene deletion character.

2. A method of producing female multiple births according to claim 1, wherein said female parent further comprises an activated SOX9 gene.

3. A method of producing female multiple births according to claim 1, wherein said female parent further comprises an activated SOX3 gene.

4. A method of producing female multiple births according to claim 1, wherein said female parent further comprises an activated DAX1 gene.

5. A method of producing female multiple births according to claim 1, wherein said female parent further comprises an activated SF1 gene.

6. A method of producing female multiple births according to claim 5, wherein said female parent further comprises a deletion of the AMH gene.

7. A method of producing female multiple births according to claim 6, wherein said female parent further comprises a deletion of the WT1 gene.

8. A method of producing female multiple births according to claim 1, wherein said female parent further comprises deletions of the DMRT1, DMRT2 and DMRT3 genes.

9. A method of producing female multiple births according to claim 1, wherein said female parent further comprises an activated WNT4 gene.

10. A method of breeding female multiple births according to any one of claims 1 to 9, wherein the multiple births trait and SRY gene deletion trait of XY chromosome female individuals expressing the multiple births trait and SRY gene deletion trait are stably inherited.